Efficient methods for multiple types of precise gene-editing in Chlamydomonas
Efficient methods for multiple types of precise gene-editing in Chlamydomonas
复制标题
DOI：
10.1111/tpj.16265
复制
发表时间：
2023-05-24
期刊：
影响因子：
7.2
通讯作者：
Huang，Kai-Yao
中科院分区：
文献类型：
--
作者：
Chen，Hui;Yang，Qing-Lin;Huang，Kai-Yao
使用CRISPR/Cas9技术进行精确的基因编辑仍然是一个长期存在的挑战，特别是对于莱茵衣藻（光合作用和纤毛研究的经典模型）中低表达和无可选择表型的基因。在这里，我们开发了一种多类型和精确的遗传操作方法，其中通过Cas9核酸酶产生DNA断裂，并使用同源DNA模板介导修复。这种方法的有效性被证明用于几种类型的基因编辑，包括两种低表达基因（CrTET 1和CrKU 80）的失活，将FLAG-HA表位标签引入VIPP 1，IFFT 46，CrTET 1和CrKU 80基因，并将YFP标签放入VIPP 1和IFFT 46用于活细胞成像。我们还成功地对FLA 3、FLA 10和FTSY基因进行了单个氨基酸替换，并记录了预期表型的获得。最后，我们证明了从MAA 7和VIPP 1的3′-UTR精确片段缺失导致稳定的敲除效应。总的来说，我们的研究为衣原体中的多种类型的精确基因编辑建立了有效的方法，能够在碱基分辨率下进行替换、插入和缺失，从而提高了这种基因编辑在基础研究和工业应用中的潜力。
Precise gene‐editing using CRISPR/Cas9 technology remains a long‐standing challenge, especially for genes with low expression and no selectable phenotypes inChlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi‐type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low‐expression genes (CrTET1andCrKU80), the introduction of aFLAG‐HAepitope tag intoVIPP1,IFT46,CrTET1andCrKU80genes, and placing aYFPtag intoVIPP1andIFT46for live‐cell imaging. We also successfully performed a single amino acid substitution for theFLA3,FLA10andFTSYgenes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3′‐UTR ofMAA7andVIPP1resulted in a stable knock‐down effect. Overall, our study has established efficient methods for multiple types of precise gene editing inChlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.